Dopamine Transporter Phosphorylation Site Threonine 53 Regulates Substrate Reuptake and Amphetamine-Stimulated Efflux

Dopamine Transporter Phosphorylation Site Threonine 53 Regulates Substrate Reuptake and Amphetamine-stimulated Efflux 2018/4/16, 3+56 PM
J Biol Chem. 2012 Aug 24; 287(35): 29702–29712. PMCID: PMC3436161

Published online 2012 Jun 21. doi: 10.1074/jbc.M112.367706 PMID: 22722938
Dopamine Transporter Phosphorylation Site Threonine 53 Regulates

Substrate Reuptake and Amphetamine-stimulated Efflux*
James D. Foster,‡,1 Jae-Won Yang,§,1 Amy E. Moritz,‡ Sathyavathi ChallaSivaKanaka,‡ Margaret A. Smith,‡
Marion Holy,§ Kyle Wilebski,‡ Harald H. Sitte,§,2,3 and Roxanne A. Vaughan‡,2,4
From the ‡Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences,
Grand Forks, North Dakota 58202-9037 and
the §Center of Physiology and Pharmacology, Institute of Pharmacology, Medical University Vienna, Waehringerstrasse 13a, A-1090
Vienna, Austria
3 To whom correspondence may be addressed. Tel.: 43-1-4277-64123; Fax: 43-1-4277-9641; E-mail: harald.sitte@meduniwien.ac.at.
4 To whom correspondence may be addressed. Tel.: 701-777-3419; Fax: 701-777-2382; E-mail: roxanne.vaughan@med.und.edu.
1Both authors contributed equally to this work.
2Both authors contributed equally to this work.
Received 2012 Mar 29; Revised 2012 Jun 13
Copyright © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
Author's Choice—Final version full access. Creative Commons Attribution Non-Commercial License applies to Author Choice Articles
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Abstract Go to:
In the central nervous system, levels of extraneuronal dopamine are controlled primarily by the action
of the dopamine transporter (DAT). Multiple signaling pathways regulate transport activity, substrate
efflux, and other DAT functions through currently unknown mechanisms. DAT is phosphorylated by
protein kinase C within a serine cluster at the distal end of the cytoplasmic N terminus, whereas recent
work in model cells revealed proline-directed phosphorylation of rat DAT at membrane-proximal
residue Thr53. In this report, we use mass spectrometry and a newly developed phospho-specific
antibody to positively identify DAT phosphorylation at Thr53 in rodent striatal tissue and heterologous
expression systems. Basal phosphorylation of Thr53 occurred with a stoichiometry of ∼50% and was
strongly increased by phorbol esters and protein phosphatase inhibitors, demonstrating modulation of
the site by signaling pathways that impact DAT activity. Mutations of Thr53 to prevent phosphorylation
led to reduced dopamine transport Vmax and total apparent loss of amphetamine-stimulated substrate
efflux, supporting a major role for this residue in the transport kinetic mechanism.
Keywords: ERK, MAP Kinases (MAPKs), Mass Spectrometry (MS), Protein Kinase C (PKC), SH3
Domains, 1-Methyl-4-phenylpyridinium (MPP+), PP1/2A, cis-trans Isomerization, Phospho-specific
Antibody, Proline-directed Phosphorylation
Introduction Go to:
The neurotransmitter dopamine (DA)5 plays a key role in many brain processes, including motor
activity, motivation, and reward. Proper dopaminergic function is dependent on the reuptake activity of
the dopamine transporter (DAT), which is the primary mechanism responsible for spatial and temporal
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436161/ Page 1 of 22
control of extraneuronal DA (1–3). Dysregulation of transport activity and consequent DA imbalance

are hypothesized to contribute to dopaminergic disorders, such as Parkinson disease, depression,
attention deficit hyperactivity disorder, and schizophrenia (4–6). DAT is also a target for many drugs of
abuse, such as cocaine and amphetamine (AMPH), and for therapeutic agents used to treat DA diseases
(7, 8). In particular, AMPH and its congeners induce multiple acute and chronic effects on DAT (9),
including reversal of transport direction (10), that lead to substrate efflux and depletion of transmitter
stores (11). The mechanism underlying efflux remains to be elucidated, but it is associated with
transporter-generated currents (12) that correlate with substrate-releasing capacity (13) and involves N-
terminal serine phosphorylation (14).
DAT is subject to extensive acute and chronic regulation that modulates DA neurotransmission in
response to momentary physiological demands and to long term disease or drug addiction states (7, 15,
16). Changes in DAT activity and cell surface expression occur in response to the actions of several
kinases and phosphatases, including protein kinase C (PKC), extracellular signal-related kinase (ERK),
and protein phosphatases 1 and 2A (PP1/2A) (17–20). DAT is phosphorylated in PKC- and
phosphatase-dependent manners, but the mechanistic relationships between transporter
phosphorylation and regulation remain unclear. Using 32PO4 metabolic labeling, we have found in rat
striatal tissue and model cells that ∼90% of rDAT phosphorylation occurs on phosphoserine (Ser(P))
and ∼10% occurs on phosphothreoine (Thr(P)) (17, 21). Phosphorylation occurs at a tonic level that is
increased with PKC activators, PP1 inhibitors, and in vitro and in vivo administration of AMPH (22),
with the majority of 32P labeling occurring in a serine cluster at the distal end of the N-terminal domain
(21–23). We recently showed that a recombinant peptide containing N-terminal residues 1–65 of rDAT
(NDAT) was phosphorylated in vitro by the proline-directed kinases ERK1/2, JNK, and p38 MAPK,
which require a proline immediately C-terminal to the phosphate acceptor (24–28). We identified the
membrane-proximal residue Thr53, which precedes Pro54, as the NDAT ERK phosphorylation site (29)
and showed the apparent total loss of Thr(P) from 32PO4 metabolically labeled rDAT carrying a Thr53
→ Ala mutation, indicating that Thr53 is a major site or the sole site of Thr(P) in the heterologously
expressed protein (29).
In this study, we use mass spectrometry and a phosphospecific antibody as positive function
approaches to demonstrate Thr53 phosphorylation of DAT and examine its characteristics without the
necessity for 32PO4 labeling or interference from PKC-induced Ser phosphorylation. Our findings
verify in vivo phosphorylation of DAT Thr53 in rat and mouse striatum as well as in heterologous
model cells and demonstrate its modulation by signaling pathways. DAT mutants containing non-
phosphorylatable residues at position 53 possessed reduced DA transport Vmax and in superfusion
assays showed complete loss of AMPH-induced substrate efflux, suggesting a crucial role for this
residue in transport kinetics. These findings reveal Thr53 phosphorylation as a novel mechanism for
regulation of DAT functions and identify the membrane-proximal region of the N terminus as a major
locus for regulation of transport kinetics.
EXPERIMENTAL PROCEDURES Go to:
Animals and Materials Protein G- and protein A-Sepharose beads were from GE Healthcare; PMA, OA,
recombinant PKCα, and ERK1 were from EMD Calbiochem; goat anti-DAT polyclonal antibody (C-
20) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Colorburst molecular mass standards,
alkaline phosphatase-linked anti-mouse and anti-rabbit IgG antibodies, and other fine chemicals were
from Sigma-Aldrich; FuGENE 6 transfection reagent and Complete Mini protease inhibitor tablets
were from Roche Applied Science; bicinchoninic acid (BCA) protein assay reagent was from Thermo
3 125
Scientific; [7,8-3H]DA (45 Ci/mmol) was from GE Healthcare; and [125I]RTI82 was synthesized and
radioiodinated as previously described (30). The recombinant NDAT was prepared and subjected to in
vitro phosphorylation with PKC and ERK1 as described previously (29). Rats were purchased from
Charles River Laboratories or the Institute for Animal Genetics, Medical University of Vienna
(Himberg), and SV129 mice were obtained from Dr. Eric Murphy (University of North Dakota). All
animals were housed and treated in accordance with regulations established by the National Institutes
of Health and approved by the University of North Dakota Institutional Animal Care and Use
Committee.
Cell Culture and DAT Mutagenesis Lewis lung carcinoma-porcine kidney (LLC-PK1) cells or LLC-PK1
cells stably expressing WT rDAT (rDAT-LLCPK1) (31) or T53A or T53D rDAT were maintained in α-
minimum essential medium supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 200 µg/ml
G418, and 100 µg/ml penicillin/streptomycin. tsA201 cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) with 10% FBS and penicillin/streptomycin. Cells were maintained in a
humidified incubation chamber gassed with 5% CO2 at 37 °C. The T53A, T53D, and T53E mutations
were made in the rDAT pcDNA 3.0 template using the Stratagene QuikChange® kit with codon
substitution verified by sequencing (Alpha Biolabs, Northwoods DNA). For production of pooled
stable transformants, transfected cells (FuGENE, Roche Applied Science) were maintained under
selection with 800 µg/ml G418 (29). tsA201 cells were transiently transfected with WT rDAT using the
ExGen500 reagent (Fermentas) according to the manufacturer's protocol. For experiments with T53E,
LLC-PK1 cells were transiently transfected with 0.6 µg of WT or T53E DNA/well using FuGENE and
assayed for [3H]DA transport activity after 24 h.
Tandem Mass Spectrometry Analysis (LC-MS/MS) Rat striatal synaptosomes, rDAT-LLCPK1 cells, or
tsA201 cells transiently expressing rDAT were solubilized in lysis buffer containing 1% Triton X-100,
20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 5 mM sodium
fluoride, 5 mM sodium pyrophosphate, and a protease inhibitor mixture (Roche Applied Science) on a
tube rotator for 2 h at 4 °C. After centrifugation at 14,000 × g for 30 min at 4 °C, the supernatant was
collected and incubated overnight with a goat anti-DAT polyclonal antibody. Immune complexes were
collected with protein G beads and washed extensively, and bound proteins were eluted in Laemmli
buffer (63 mM Tris-HCl, 10% glycerol, 2% SDS, 3% 2-mercaptoethanol, 100 mM dithiothreitol,
0.0025% bromphenol blue, pH 6.8) at 95 °C for 3 min. Eluted proteins were size-fractionated on SDS-
polyacrylamide gels and visualized by Coomassie Brilliant Blue staining, and the indicated band was
excised. Gel pieces were destained with 50% acetonitrile in 25 mM ammonium bicarbonate and dried in
a speed vacuum concentrator. After reduction and alkylation of cysteine residues, gel pieces were
washed and dehydrated. Dried gel pieces were rehydrated with 25 mM ammonium bicarbonate (pH 8.0)
containing 10 ng/µl trypsin or chymotrypsin (Promega, Madison, WI) and incubated for 18 h at 37 °C.
The digested peptide mixtures were extracted with 50% acetonitrile in 5% formic acid and
concentrated in a speed vacuum concentrator for LC-MS/MS. An ion trap mass spectrometer (HCT,
BrukerDaltonics, Bremen, Germany) coupled with an Ultimate 3000 nano-HPLC system (Dionex,
Sunnyvale, CA) was used for LC-MS/MS data acquisition. A PepMap100 C-18 trap column (300 µm ×
5 mm) and PepMap100 C-18 analytic column (75 µm × 150 mm) were used for reverse phase
chromatographic separation with a flow rate of 300 nl/min. The two buffers used for the reverse phase
chromatography were 0.1% formic acid, water (buffer A) and 0.08% formic acid, acetonitrile (buffer
B) with a 125 min gradient (4–30% B for 105 min, 80% B for 5 min, and 4% B for 15 min). Eluted
peptides were then directly sprayed into the mass spectrometer to record peptide spectra over the mass
range of m/z 350–1500 and MS/MS spectra in information-dependent data acquisition over the mass
range of m/z 100–2800. Repeatedly, MS spectra were recorded, followed by three data-dependent
collision-induced dissociation MS/MS spectra generated from the four highest intensity precursor ions.
The MS/MS spectra were interpreted with the Mascot search engine (Matrix Science, London, UK).
Data base searches through Mascot were performed with a mass tolerance of 0.5 Da and an MS/MS
tolerance of 0.5 Da; three missing cleavage sites and carbamidomethylation on cysteine, oxidation on
methionine, deamidation on asparagine/glutamine, and phosphorylation on serine/threonine were
allowed. Each filtered MS/MS spectra exhibiting possible phosphorylation was manually checked and
validated (32, 33).
Phospho-specific Antibody Generation A Threonine 53-phosphospecific polyclonal antibody (Thr(P)53

Ab) against DAT was generated by PhosphoSolutions (Aurora, CO). Briefly, rabbits were immunized
with a phosphopeptide based on the DAT N-terminal amino acid sequence:
TNSTLINPPQpTPVEAQERTW (Thr(P)53 shown in boldface type). Control peptide consisting of the
identical sequence with non-phosphorylated Thr53 was also synthesized. Thr(P)53-specific polyclonal
antibody present in immune serum was purified through sequential rounds of chromatography against
immobilized phospho- and dephosphopeptide and concentrated to 1 mg/ml. Affinity-purified antibody
screened by ELISA showed strong reactivity against the Thr(P)53 peptide antigen and essentially no
reactivity to the corresponding dephosphopeptide (not shown).
DAT Immunoblot and Immunoprecipitation Immunoblotting was performed with mouse monoclonal N-
terminal Ab 16 (mAb 16) generated against residues 42–59 to detect total DAT as described previously
(34) or with rabbit polyclonal Thr(P)53 Ab generated in this study. Briefly, lysates of rodent striatal
membranes or rDAT-LLCPK1 cells were resolved on 4–20% SDS-polyacrylamide gels using
ColorBurst (Sigma) molecular mass markers as standards. For regulation studies, rat striatal
synaptosomes were prepared and treated with vehicle, phorbol 12-myristate 13-acetate (PMA), or
okadaic acid (OA) for 30 min at 30 °C as described previously (35), followed by lysis and
electrophoresis. Gels were transferred to PVDF and blocked, followed by incubation with primary
antibodies used at 1:1000 dilutions. Where indicated, N-terminal peptides with or without Thr53
phosphorylation were included with the primary antibodies at 50 µg/ml. Immunostaining was detected
using anti-mouse or anti-rabbit alkaline phosphatase-conjugated secondary antibodies and
chemiluminescent light detection using ImmunStar (Bio-Rad) substrate and a Bio-Rad gel
documentation system. For quantification of Thr53 phosphorylation, Thr(P)53 Ab staining was
normalized to total DAT levels determined in parallel using mAb 16, and statistical analysis was
performed using ANOVA. For immunoprecipitation studies, lysates of unlabeled or [125I]RTI82-
labeled rat striatal membranes (36) were immunoprecipitated with polyclonal Ab 16 or Thr(P)53 Ab (3
µg) using procedures previously described (37). Precipitated DATs were resolved on 4–20% SDS-
polyacrylamide gels and were transferred for subsequent immunoblotting or were dried and exposed to
x-ray film for 3–7 days at −80 °C.
Determination of Thr53 Phosphorylation Stoichiometry Rat striatal lysates were immunoprecipitated with
Thr(P)53 Ab, and bound and unbound fractions were immunoblotted with Thr(P)53 Ab to determine the
fraction of Thr53-phosphorylated transporters retained in the pellet. Bound samples were also blotted
with mAb 16 to detect total DAT protein in the pellet, and the fraction of input DAT pulled down by
Thr(P)53 Ab was determined by comparing the staining intensities of the Thr(P)53 Ab pellet with that
of a DAT standard curve generated by titration of input sample and immunoblotted in parallel with
mAb 16. The Thr(P)53 stoichiometry estimate was determined by dividing the fraction of input DAT
present in the Thr(P)53 Ab pellets by the Thr(P)53 Ab precipitation efficiency.
DA Uptake and Cell Surface Biotinylation Assays WT or mutant rDAT-LLCPK1 cells were grown in 24-
well plates to 70–80% confluence in α-minimum essential medium at 37 °C. Cells were rinsed twice
with 0.5 ml of Krebs-Ringer-HEPES (KRH) buffer (25 mM HEPES, 125 mM NaCl, 4.8 mM KCl, 1.2
mM KH2PO4, 1.3 mM CaCl2, 1.2 mM MgSO4, 5.6 mM glucose, pH 7.4) followed by the addition of 0.5
ml of warmed KRH buffer (37 °C) and uptake assay. Uptake was performed in triplicate and initiated
by the addition of 10 nM [3H]DA plus 0.3–30 µM unlabeled DA (where indicated). Nonspecific uptake
was determined in the presence of 100 µM (−)-cocaine. Uptake was allowed to proceed for 8 min at 37
°C, and cells were rapidly washed three times with ice-cold KRH buffer. Cells were solubilized in 1%
Triton X-100, radioactivity contained in lysates was assessed by liquid scintillation counting, and
protein content was assessed using BCA colorimetric reagent. For cell surface expression
determination, WT or mutant rDAT-LLCPK1 cells were incubated with the membrane-impermeable
biotinylating reagent sulfosuccinimidyl-2[biotinamido]ethyl-1,3-dithiopropionate sulfo-NHS-SS-
biotin, and biotinylated DATs were purified from cell lysates (25 µg of protein) by chromatography on
NeutrAvidin beads, separated by SDS-PAGE, and quantified by immunoblotting (38). For ion dose-
response experiments, Na+ and Cl− were replaced across the range of 0–150 mM with N-methyl D-
gluconate or sodium acetate, respectively, and uptake was analyzed using 10 nM [3H]DA plus 3 µM DA
(39, 40).
Superfusion Experiments Substrate efflux assays were performed as previously described (41). In brief,
culture medium was removed from stably transfected WT or mutant rDAT LLC-PK1 cells (see above;
4 × 105 cells/well grown on coverslips in 96-well plates) and exchanged with KRH buffer. In all
superfusion assays, we used [3H]1-methyl-4-phenylpyridinium (MPP+) (85 Ci/mmol; American
Radiolabeled Chemicals, St. Louis, MO) as the DAT substrate because it is metabolically inert, cannot
diffuse out of the cells, and thereby significantly enhances the signal/noise ratio of the experiment (42).
The cells were preincubated with 0.1 µM MPP+ for 20 min at 37 °C in a final volume of 0.1 ml of KRH
buffer/well and subsequently transferred into superfusion chambers. Immediately, superfusion was
initiated with KRH buffer at 25 °C at a perfusion rate of 0.7 ml/min. After 45 min, a stable efflux of
radioactivity was achieved, and the experiment was started with the collection of 2-min fractions. After
three fractions, AMPH (3 µM) was added to stimulate the reverse operation of DAT.
RESULTS Go to:
Identification of Phosphorylated Thr53 on DAT by Mass Spectrometry To identify in vivo DAT

phosphorylation, we immunopurified DAT from rat striatal lysates and size-fractionated purified
proteins by SDS-PAGE (Fig. 1A). The indicated Coomassie Blue-stained band was subjected to trypsin
in-gel digestion, and resulting peptides were subjected to liquid chromatography tandem MS (LC-
MS/MS). LC-MS/MS identified DAT protein (Swiss-Prot ID: P23977) with 97 matched peptides and
30.2% sequence coverage (Fig. 1B, top). Thr(P) was unambiguously and repeatedly identified at
residue 53 in the MS/MS spectra of the tryptic peptide spanning amino acid residues 30–60
(EQNGVQLTNSTLINPPQpTPVEAQER) (Fig. 1C). To confirm phosphorylation of DAT at Thr53 in
heterologous cells, we also analyzed rDAT transiently or stably expressed in mammalian cell lines.
Using the same MS approach, we found that DAT was phosphorylated at Thr53 in both tsA201 and
rDAT-LLCPK1 cell lines (Fig. 1D). The use of chymotrypsin for in-gel digestion substantially
increased sequence coverage. MS/MS analysis mapped rDAT protein with individual sequence
coverage of 30.7 and 52.7% for tryptic and chymotryptic peptides, respectively (data not shown),
summing up to a total sequence coverage of 68.7% of the rDAT protein in rDAT-LLCPK1 cells with
Thr(P)53 (Fig. 1B, bottom).
Open in a separate window

FIGURE 1.
Identification of Thr53 phosphorylation of DAT in rat striatum and heterologous cells by LC-MS/MS.
A, Coomassie Blue-stained SDS-PAGE gel with immunopurified rat striatal DAT (arrow). Molecular mass
markers are indicated on the left. B, sequence coverage of DAT with identified peptides (boldface type) by
MS/MS from rat striatum (top) and rDAT-LLCPK1 cells (bottom). The putative 12 transmembrane segments
(S1–S12) of DAT are underlined, and the identified phosphorylation site at Thr53 is indicated by a box. C, the
spectrum of triple charged rDAT peptide obtained at m/z 948.45 was fragmented to produce a tandem mass
spectrum with y- and b-ion series. The MS/MS spectrum shows phosphorylated Threonine (pT) in the
sequence EQNGVQLTNSTLINPPQpTPVEAQER (amino acids 36–60). D, MS/MS spectrum from rDAT
transiently expressed in tsA201 cells (top) and stably expressed in LLC-PK1 cells (bottom). The triple
charged, tryptic peptide at m/z 948.44 from heterologous tsA201 cells was fragmented to y- and b-ion series
that described the sequence EQNGVQLTNSTLINPPQpTPVEAQER with phosphorylation at Thr53. In rDAT-
LLCPK1 cells, the spectrum of double charged, chymotryptic peptide at m/z 987.99 presents the unambiguous
identification of Thr(P)53 in the sequence INPPQpTPVEAQERETW of rDAT.
53
Characterization of Thr(P)53 Ab Immunoreactivity We then assessed the ability of our phospho-specific

53
antibody to detect Thr(P) in the DAT sequence. In our previous study (29), we demonstrated that the
recombinant N-terminal domain peptide NDAT was phosphorylated in vitro by ERK1/2 at Thr53,
whereas more recently we have determined that PKC-catalyzed phosphorylation of NDAT occurs on
multiple sites in the distal serine cluster, paralleling PKC-induced metabolic phosphorylation of this
domain (21) (Fig. 2A). We used these differentially phosphorylated NDAT samples to test the
specificity of the Thr(P)53 Ab in immunoblot assays. Thr(P)53 Ab did not recognize NDAT that was
not phosphorylated or was phosphorylated by PKC, but it was strongly reactive against ERK-
phosphorylated NDAT (Fig. 2B, top). Staining of ERK-phosphorylated NDAT was lost when Thr(P)53
Ab was preincubated with Thr(P)53 peptide but was not affected by incubation with the corresponding
dephosphopeptide (Fig. 2B, top). Staining of blots with mAb 16 verifies equal NDAT protein in all
samples (Fig. 2B, bottom) and demonstrates the upward shift of NDAT induced by Thr53
phosphorylation that we previously reported (29). These results demonstrate that Thr(P)53 Ab
specifically recognizes DAT N-terminal tail sequence phosphorylated at Thr53 and does not recognize
N-terminal sequence that is not phosphorylated or is phosphorylated by PKC on distal serines.

FIGURE 2.
53
Immunoreactivity of NDAT with Thr(P)53 Ab. A, rat DAT N-terminal amino acid sequence (residues 1–65;
NDAT) highlighting the PKC-dependent phosphorylation domain (PKC) and the ERK proline-dependent
phosphorylation site (ERK). B, NDAT samples given no kinase treatments or phosphorylated in vitro by
PKCα or ERK1 were immunoblotted with Thr(P)53 Ab (pT53Ab) (top) or mAb 16 (bottom). Where indicated,
Thr(P)53 Ab was preincubated with phosphorylated (pT53) or non-phosphorylated (Ctl) N-terminal peptide.
Molecular mass markers are indicated on the right.
Thr(P)53 Ab Staining of Expressed DAT We then assessed the ability of Thr(P)53 Ab to recognize DAT
expressed heterologously in LLCPK1 cells (Fig. 3A). Thr(P)53 Ab produced little or no
immunostaining of lysates from LLCPK1 cells but showed strong immunoreactivity against a ∼90 kDa
band in rDAT-LLCPK1 lysates, indicating specific recognition of rDAT (Fig. 3A, upper panels).
Importantly, Thr(P)53 Ab showed no reactivity against T53A rDAT (Fig. 3A, left), and immunostaining
of WT DAT was prevented by inclusion of Thr(P)53 peptide but not by the corresponding
dephosphopeptide (Fig. 3A, right). Equal levels of WT and T53A rDAT protein and the absence of
rDAT in LLCPK1 cells are shown by mAb 16 staining (Fig. 3A, bottom panels). These results
demonstrate high specificity of the Thr(P)53 Ab toward phosphorylated Thr53 of rDAT, confirming the
mass spectrometry results and supporting our previous loss-of-function evidence for Thr53
phosphorylation in expressed DAT (29).

FIGURE 3.
Immunoreactivity of DAT with Thr(P)53 Ab. Lysates from LLC-PK1 cells, rDAT-LLCPK1 cells, or T53A
rDAT-LLCPK1 cells (A) or lysates from rDAT-LLCPK1 cells (B) were immunoblotted with Tyr(P)53 Ab
(pT53Ab) (top panels) or mAb 16 (bottom panels). Where indicated, Thr(P)53 Ab was preincubated with
phosphorylated (pT53) or non-phosphorylated (Ctl) N-terminal peptide. Molecular mass markers are
indicated on the right. C and D, lysates from rat or mouse striatal or rat cerebellar (CB) tissue were
immunoblotted with Thr(P)53 Ab (top panels) or mAb 16 (bottom panels). Where indicated, Thr(P)53 Ab was
preincubated with phosphorylated (pT53) or non-phosphorylated (Ctl) N-terminal peptide. Molecular mass
markers are indicated on the right. E, rat striatal membranes with (top and middle panels) or without (bottom
panel) [125I]RTI-82 photoaffinity labeling of DAT were immunoprecipitated (IP) with Thr(P)53 Ab or
polyclonal Ab 16 as indicated and resolved by SDS-PAGE. Gels were dried and subjected to autoradiography
(top and middle panels) or transferred to PVDF and probed with mAb 16 (bottom panels). Where indicated,
Thr(P)53 Ab was preincubated with phosphorylated (pT53) or non-phosphorylated (Ctl) N-terminal peptide.
53
We also noted in these experiments that Thr(P)53 Ab stained the unglycosylated 60-kDa form of DAT
as well as the fully glycosylated 90-kDa form (Fig. 3A, right). The ratio of mature and immature form
staining by Thr(P)53 Ab was comparable with that detected by mAb 16, indicating that both forms
possess similar levels of Thr53 phosphorylation. We also frequently observed a minor band at >100
kDa in parent cell and T53A rDAT lysates (Fig. 3A, left), indicating the presence of a small degree of
cross-reactivity of this Ab with another protein. However the intensity of this staining is negligible in
comparison with that of DAT.
Thr(P)53 Ab Staining of Rat and Mouse Striatal DAT Next we immunoblotted rat and mouse striatal DAT
with Thr(P)53 Ab (Fig. 3B). Thr(P)53 Ab was highly reactive against a protein of ∼90 kDa from rat
striatal tissue, with only negligible staining detected in cerebellar tissue, which does not express DAT (
Fig. 3B, left), strongly supporting the identity of the band as DAT. Staining of the rat striatal band was
blocked by inclusion of Thr(P)53 peptide but not by the dephosphopeptide (Fig. 3B, right),
demonstrating Ab specificity for phosphorylated Thr53. Levels of DAT protein in each lane are
indicated by mAb 16 staining (bottom panels). Mouse DAT, which possesses the Thr53-Pro54 sequence
(43), also showed strong immunoreactivity with Thr(P)53 Ab (Fig. 3B, left), whereas human DAT,
which possesses the sequence Ser53-Pro54 (44), did not show Thr(P)53 Ab immunostaining (not
shown), further supporting the Ab specificity for Thr(P)53. Staining of a minor band in the cerebellar
sample in the absence of DAT (Fig. 3B, right) further indicates a slight reactivity of this Ab with a
different protein.
Immunoprecipitation of Rat Striatal DAT with Thr(P)53 Ab We also demonstrated the ability of Thr(P)53
Ab to immunoprecipitate DAT (Fig. 3C). Thr(P)53 Ab readily precipitated rat striatal DATs
photoaffinity-labeled with the cocaine analog [125I]RTI-82 (Fig. 3C, top) to levels that were
comparable with that precipitated using our standard procedures with polyclonal Ab 16 (Fig. 3C,
middle). Non-photolabeled rDATs precipitated by Thr(P)53 Ab could also be detected by
immunoblotting with mAb 16 (Fig. 3C, bottom). In both cases, Thr(P)53 Ab-mediated precipitation
was blocked with Thr(P)53 peptide but not with the dephosphopeptide (Fig. 3C, top), demonstrating
specificity for Thr53 phosphorylation.
Modulation of Thr53 DAT Phosphorylation DAT phosphorylation has been studied primarily by metabolic
labeling with 32PO4 and has been demonstrated to be modulated by PKC, AMPH, and protein
phosphatases (17, 21, 22, 45–47). However, the vast majority of basal and stimulated 32PO4 labeling
occurs on distal N-terminal serines (21), making this method unfeasible for characterization of Thr53
phosphorylation responses. To examine the potential for Thr(P)53 Ab to detect regulation of Thr53
phosphorylation, rDAT-LLCPK1 cells and rat striatal tissue were treated with OA to inhibit protein
phosphatases or with PMA to activate PKC, followed by blotting with Thr(P)53 Ab or mAb 16. OA and
PMA treatments caused Thr53 phosphorylation to increase to 178 ± 20% and 140 ± 13% of basal levels
(p < 0.01 and p < 0.05, respectively) in rDAT-LLCPK1 cells (Fig. 4A) and to 174 ± 15% and 168 ±
23% of basal levels (p < 0.001 and p < 0.05, respectively) in rat striatal synaptosomes (Fig. 4B). These
results show the ability of Thr(P)53 Ab to detect changes in Thr53 phosphorylation levels and
demonstrate the acute modulation of rDAT Thr53 phosphorylation by PKC and phosphatase pathways
in both expression systems and native tissue.

FIGURE 4.
Regulation of Thr53 phosphorylation by phorbol ester and okadaic acid. A, rDAT-LLCPK1 cells were
treated with vehicle, 1 µM OA, or 1 µM PMA for 30 min at 37 °C. B, rat striatal synaptosomes were treated
with vehicle, 1 µM OA, or 1 µM PMA for 30 min at 30 °C. Lysates were subjected to SDS-PAGE and
immunoblotted with Thr(P)53 Ab (pT53Ab) (top) or mAb 16 (bottom) (representative examples shown).
Histograms show quantification of Thr(P)53 staining relative to vehicle controls (means ± S.E.; n = 3) (*, p <
0.05; **, p < 0.01; ***, p < 0.001 relative to basal by ANOVA).
Phosphorylation Stoichiometry To obtain an estimate of basal phosphorylation stoichiometry, we

53 53
analyzed lysates of untreated rat striatal tissue. Thr(P) Ab was used to specifically extract Thr -
phosphorylated protein in immunoprecipitation procedures. Analysis of the bound and unbound
fractions by Thr(P)53 Ab immunoblotting showed that Thr53-phosphorylated DATs were captured with
an efficiency of 38 ± 1%. The amount of total DAT in the precipitated sample was estimated to be 20 ±
1% of the input value, as determined by comparison of mAb 16 staining of Thr(P)53 Ab pellets with
DAT standard curves generated by dilutions of input striatal lysate and immunoblotted in parallel with
mAb 16. Normalizing the fraction of DAT protein in the Thr(P)53 Ab pellet by the Thr(P)53 Ab
precipitation efficiency yielded a Thr53 phosphorylation stoichiometry estimate of 53 ± 2% (data not
shown; all values means ± S.E. from three independent experiments performed in duplicate).
53
Kinetic Analysis of Thr53 Mutants To examine possible functions of Thr phosphorylation, we
generated T53A non-phosphorylatable and T53D phosphomimetic mutants for stable expression in
LLCPK1 cells. mAb 16 immunoblotting showed that T53A and T53D mutants co-migrated with the
WT protein at ∼90 kDa on SDS-polyacrylamide gels, indicating full glycosylation and proper
biosynthetic processing (48). Total expression levels of the mutants were 61 ± 7% and 75 ± 7% of the
WT protein level (Fig. 5A), and plasma membrane levels assessed by cell surface biotinylation were
directly proportional to total expression (60 ± 10% and 71 ± 6% of WT surface level) (Fig. 5B),
indicating no significant impacts of the mutations on trafficking or surface presentation.

FIGURE 5.
Expression and kinetic analysis of T53A and T53D DATs. rDAT-LLCPK1 cells stably expressing WT,
T53A, or T53D DATs were assayed in parallel for total DAT expression levels (A), surface DAT expression
levels (B), and DA uptake saturation analysis (C). Blots shown are representative of four independent
experiments with samples run in duplicate. Histograms show quantification of DAT levels (**, p < 0.01; *, p
< 0.05 relative to controls by ANOVA with Tukey's post hoc test). Transport kinetic parameters were
determined by nonlinear regression analysis in three or four independent experiments performed in triplicate.
Data are presented as means ± S.E. normalized to cell surface DAT expression levels (**, p < 0.01; *, p <
0.05 relative to controls by ANOVA with Tukey's post hoc test).
Saturation analyses (Fig. 5C) showed that after normalizing DA transport for relative DAT plasma
membrane expression, both mutants possessed significantly lower Vmax values than the WT protein
(WT, 925 ± 34 pmol/min/mg; T53A, 588 ± 53 pmol/min/mg, p < 0.01; T53D, 664 ± 53 pmol/min/mg,
p < 0.05; ANOVA). DA Km values were not different (WT, 2.0 ± 0.2 µM; T53A, 1.8 ± 0.4 µM; T53D,
1.6 ± 0.1 µM; p > 0.05), implicating alteration of transport turnover rate rather than DA recognition as
the mechanism for reduced transport in the mutants. To determine if glutamic acid mutation of Thr53
would provide a superior phosphomimetic substitution, we generated T53E DAT for analysis in
transiently transfected cells. T53E DAT showed comparable expression relative to the WT protein in
mAb 16 immunoblots, but in saturation analyses, it showed DA transport Vmax reductions that were
similar in magnitude to that of T53D DAT (not shown), indicating that neither phosphomimetic
substitution could rescue the transport reduction seen with the T53A mutation.
To determine if alterations in ion interactions could underlie the reduced Vmax values obtained with
Thr53 mutation, we analyzed WT and T53A proteins for Na+ and Cl− concentration dependence. In
three independent experiments, we found no significant differences in EC50 of DA uptake for Na+
(WT, 84.4 ± 1.5 mM; T53A, 84.3 ± 4.2 mM; p > 0.05) or for Cl− (WT, 58.9 ± 4.6 mM; T53A, 58.5 ± 1.8
mM; p > 0.05), indicating that loss of Thr53 did not lead to reduced uptake via impacts on the ability of
Na+ or Cl− to drive DA translocation.
We then used whole-cell superfusion assays to examine the mutants for AMPH-stimulated substrate
efflux using [3H]MPP+ as the substrate. MPP+ was robustly transported by the mutants (Vmax WT, 141
± 7 pmol/min/mg; T53A, 97 ± 14 pmol/min/mg; T53D, 105 ± 12 pmol/min/mg, p > 0.05, n = 3),
allowing adequate loading of substrate for efflux analysis. Base-line efflux quantified as a fraction of
intracellular substrate did not differ between cell lines (Fig. 6). However, although application of
AMPH induced robust MPP+ efflux in the WT rDAT-LLCPK1 cells, it produced no detectable
substrate release in the rDAT-T53A and T53D cells (Fig. 6). Lack of AMPH-stimulated efflux activity
in the mutants is not due to inability to recognize AMPH because 3 µM AMPH inhibited [3H]DA
uptake by 75–85% in all three cell lines (not shown). These results suggest that Thr53 and/or its ability
to undergo phosphorylation/dephosphorylation exert a mechanistic role in inward DA transport and
constitute a major prerequisite for AMPH-mediated MPP+ efflux.

FIGURE 6.
Amphetamine-stimulated efflux of T53A and T53D DATs. LLC-PK1 cells stably expressing WT, T53A, or
T53D DATs were preloaded with [3H]MPP+ for 20 min at 37 °C followed by superfusion analysis of efflux as
described under “Experimental Procedures.” Upon reaching a stable base line, three 2-min fractions were
collected to define basal efflux followed by the addition of 3 µM AMPH (arrow) to stimulate DAT-mediated
efflux.
DISCUSSION Go to:
In this study, we use mass spectrometry and phospho-specific immunostaining as positive function
approaches to identify and characterize phosphorylation of DAT Thr53 in rodent striatum and
heterologous cells. Thr53 is present in the membrane-proximal region of the N-terminal tail close to the
beginning of transmembrane domain 1 (TM1) in a motif specific for proline-directed kinases, such as
ERK1/2, p38 MAPK, and JNK (29). Although we do not yet know which kinase(s) phosphorylate
Thr53, our results provide the first evidence that DAT is directly acted on in neurons by this class of
enzymes.
The proline-directed kinase with the most well documented effects on DAT is ERK. In cells and striatal
tissue, ERK inhibitors induce rapid reductions in DA transport activity (16, 20, 49, 50), whereas
activation of D2 and D3 DA receptors induces ERK-dependent up-regulation of DAT (7, 51). These
findings indicate that DA transport functionality is positively influenced by tonic ERK signaling and
that rapid up-regulation of transport capacity can be achieved by receptor-mediated activation of ERK.
Thus, our findings supporting involvement of Thr53 in forward and reverse transport suggest a
mechanistic basis for regulation of DAT by ERK. Although we previously found no loss of ERK
inhibitor effects on uptake activity of T53A DAT (29), the reduced DA transport Vmax we found in
T53A and T53D mutants is consistent with a role for ERK-mediated phosphorylation of Thr53 in
maintaining transport activity. If future studies support this idea for ERK or related kinases, our
estimate that basal phosphorylation of Thr53 occurs with a stoichiometry of ∼50% suggests a tonic set
point readily amenable to either increased or decreased phosphorylation of significant DAT copy
numbers to allow robust modulation of transport capacity. Other potential roles for this site could
include tyrosine kinase regulation of DAT, which also involves MAPK activity (52), or ERK-
associated functions in cocaine and AMPH actions (3, 53). Current efforts are under way in our
laboratory to test Thr53 phosphorylation in modulating ERK effects on DAT and to investigate
potential regulatory inputs from other proline-directed kinases.
Our results also strongly support a role for Thr53 phosphorylation in the mechanism of AMPH-
stimulated efflux, which is considered to be a crucial factor in the reinforcing and neurotoxic properties
of AMPH and related drugs (54). The finding that Thr53 exerts a mechanistic role in efflux is striking
because the involvement of phosphorylation in reverse transport has previously been attributed to distal
N-terminal serines (55). At present, we lack a clear understanding of the relative contributions of these
two N-terminal regions in the efflux mechanism and do not know if there is communication between
the domains with respect to phosphorylation. However, the importance of the membrane-proximal
region of the N terminus in uptake and efflux mechanisms has been shown in several recent
mutagenesis studies (6, 56, 57). In particular, the efflux properties of T53A and T53D mutants
resemble those found for a SERT construct with the N terminus tethered to the plasma membrane by
TAC peptide (56), supporting a crucial role for the precise arrangement and conformational flexibility
of the N terminus in the mechanism of transport reversal.
Proline-directed phosphorylation is well established to drive major protein structural rearrangements by

inducing cis-isomerization of the peptide backbone around the Ser(P)/Thr(P)–Pro bond (24, 25). This is
likely to be the case for DAT as well because ERK phosphorylation of NDAT causes a distinct upward
shift in electrophoretic mobility that is not induced by PKC or other AGC kinases (29). Upward shifts
in DAT mobility on SDS-polyacrylamide gels induced by phosphorylation conditions have also been
noted (16, 45, 58), consistent with altered conformations that could result from Thr53-Pro54 cis-
isomerization. Phosphorylation of Thr53 is thus likely to play a major role in directing the
conformational state of the membrane-proximal region of the DAT N terminus.
Our findings that T53A and T53D mutants showed reduced Vmax for forward DA transport and
complete loss of AMPH-stimulated reverse MPP+ transport strongly support a crucial role for Thr53 in
the transport mechanism. TM1 of DAT performs an essential role in substrate transport and
psychostimulant drug binding (8), and the proximity of Thr53 to TM1 suggests the potential for its
phosphorylation and/or Thr53-Pro54 cis-trans isomerization to impact transport kinetics via effects on
TM1 conformation. Alternatively, Thr53 phosphorylation and/or Thr53-Pro54 cis-trans isomerization
could potentially impact the ability of nearby intracellular gate residue Arg60 to form a salt bridge with
Asp436 (59), which could affect molecular transitions necessary for the transport cycle. At present, we
cannot distinguish between Thr53 contributions to uptake and efflux as due to side chain hydrogen
bonding, direct phosphorylation, or peptide backbone cis-trans isomerization. However, if an inability
to undergo phosphorylation underlies the transport and efflux reductions seen in T53A DAT, the
putative phosphomimetic T53D and T53E mutations do not satisfy the requirements necessary to
support these functions. This is probably due to the inability of Asp or Glu to fully compensate for
phosphoryl group charge interactions or to induce backbone cis-isomerization.
53
The proline-rich sequence immediately surrounding Thr53 (PPQTP) also constitutes an Src homology 3
(SH3) domain epitope (PXXP) (60) that may serve as a ligand for interactions with SH3 domain
proteins. This motif may therefore function to direct DAT oligomer formation or DAT-binding partner
interactions (2, 61). Oligomerization is the preferred quaternary state of neurotransmitter transporters
(62) and has been shown to play an important role in transporter-mediated efflux (63). DAT
oligomerization is also regulated by AMPH (64), suggesting the possibility that AMPH-mediated
activation of PKC (65) may regulate DAT monomer-oligomer equilibria through effects on Thr53
phosphorylation. In addition, several proteins that affect DA transport and efflux, including syntaxin
1A and receptor for activated protein kinase C1, interact with the DAT N-terminal domain (66–68),
suggesting them as possible intermediaries for Thr53 effects.
We found strongly increased phosphorylation of DAT Thr53 in rat striatal synaptosomes and cells
treated with OA, indicating the presence of robust protein phosphatase activity that maintains this
residue in the dephosphorylated state (46). The dose of OA used (10 µM) is compatible with inhibition
of PP2A and PP1, both of which have been associated with DAT (46, 69). Thr53 phosphorylation is
also stimulated by PMA; however, PKC cannot directly phosphorylate proline-directed residues (70)
and does not phosphorylate NDAT on Thr53 in vitro (29). Thus, it is likely that stimulation of Thr53
phosphorylation by PMA occurs via PKC cross-talk with pathways for ERK or other proline-dependent
kinases (71–76). These findings thus demonstrate the ability of Thr53 phosphorylation to be regulated
either directly via proline-directed kinases and phosphatases or indirectly through PKC modulation of
downstream pathways, indicating the potential for Thr53 to serve as a locus for integration of DAT
regulatory signals. In addition, both OA and PMA induce DAT down-regulation and endocytosis (16),
supporting the potential for Thr53 phosphorylation to participate in these processes.
Although the structure of the DAT N terminus is unknown, the sequence consists of two Ser/Thr-rich
domains in membrane-distal (amino acids 1–21) and membrane-proximal (amino acids 43–64) regions,
separated by a charged/hydrophobic sequence devoid of potential phosphate acceptors (amino acids
22–42) (Fig. 2). We have now demonstrated that DAT is phosphorylated by PKC in the membrane-
distal region and by proline-directed kinases in the membrane-proximal region (21, 29). Because PKC
and other AGC kinases do not act on Ser/Thr residues that precede proline (27, 70, 77), and none of the
distal N-terminal serines are present in proline-directed motifs, our findings provide direct evidence
that the proximal and distal phosphorylation domains are acted on by functionally distinct classes of
protein kinases. This suggests a separation of N-terminal functional mechanisms that may differentially
impact transporter regulation and present potential sites for therapeutic modulation of transport activity
in dopaminergic diseases.
Acknowledgments Go to:
We thank Drs. Amy Newman and John Lever for supplying [125I]RTI82, Dr. Eric Murphy for
supplying SV129 mice, and Drs. Gert Lubec and Wei-Qiang Chen for generous support with mass
spectrometry.
*This work was supported, in whole or in part, by National Institutes of Health, Grant R01 DA13147 from NIDA (to
R. A. V.), ND EPSCoR IIG (to R. A. V. and J. D. F.), P20 RR017699 from the COBRE program of the National
Center for Research Resources (to the University of North Dakota), and P20 RR016741 from the IDeA Networks of
Biomedical Research Excellence (INBRE) program of the National Center for Research Resources (to the
University of North Dakota). This work was also supported by Austrian Research Funds/FWF Grants F3506 and
P22893-B1 (to H. H. S.) and P23670-B09 (to J.-W. Y.).
5The abbreviations are:
DA dopamine
DAT dopamine transporter
PMA phorbol 12-myristate 13-acetate
OA okadaic acid
AMPH amphetamine
MPP+ [3H]1-methyl-4-phenylpyridinium
NDAT recombinant DAT N-terminal tail protein
rDAT rat DAT
SH3 Src homology 3
TM1 transmembrane domain 1
Ab antibody
ANOVA analysis of variance.
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Dopamine Transporter Phosphorylation Site Threonine 53 Regulates Substrate Reuptake and Amphetamine-Stimulated Efflux

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Dopamine Transporter Phosphorylation Site Threonine 53 Regulates Substrate Reuptake and Amphetamine-Stimulated Efflux

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Dopamine Transporter Phosphorylation Site Threonine 53 Regulates Substrate Reuptake and Amphetamine-stimulated Efflux 2018/4/16, 3+56 PM

J Biol Chem. 2012 Aug 24; 287(35): 29702–29712. PMCID: PMC3436161

Dopamine Transporter Phosphorylation Site Threonine 53 Regulates

Received 2012 Mar 29; Revised 2012 Jun 13

This article has been cited by other articles in PMC.

control of extraneuronal DA (1–3). Dysregulation of transport activity and consequent DA imbalance

EXPERIMENTAL PROCEDURES Go to:

Phospho-specific Antibody Generation A Threonine 53-phosphospecific polyclonal antibody (Thr(P)53

Identification of Phosphorylated Thr53 on DAT by Mass Spectrometry To identify in vivo DAT

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Characterization of Thr(P)53 Ab Immunoreactivity We then assessed the ability of our phospho-specific

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Phosphorylation Stoichiometry To obtain an estimate of basal phosphorylation stoichiometry, we

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Proline-directed phosphorylation is well established to drive major protein structural rearrangements by

5The abbreviations are:

45. Vaughan R. A. (2004) Phosphorylation and regulation of psychostimulant-sensitive

628–649 [PMC free article] [PubMed]

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